DESCRIPTION: (Investigator's abstract) This is the competitive renewal of a grant funded approximately three years ago to develop a mouse model of sickle cell disease. During the funding period we produced transgenic mice that switch from human fetal hemoglobin to human sickle hemoglobin around three weeks after birth. These animals were bred with our beta-thalassemic mice containing a knockout mutation of the beta-maj and beta-min globin genes and with Paszty et al's alpha-thalassemic mice containing a knockout mutation of the alphal and alpha2 globin genes. The resulting animals synthesize only human hemoglobin in adult red blood cells. Similar to many human patients with sickle cell disease, the mice develop a severe hemolytic anemia and extensive organ pathology. Numerous sickled erythrocytes are observed in peripheral blood. Although chronically anemic, most animals survive for 2 to 9 months and are fertile. We now propose to correct sickle cell disease in these mice by transduction of hematopoietic stem cells with viral vectors containing anti-sickling genes under control of human beta-globin Locus Control Region (LCR) sequences. One limitation in this strategy has been the inefficiency of transduction into quiescent stem cells. We have now demonstrated that Sca-1+, c-Kit+, Lin- bone marrow stem cells that are isolated without cytokine prestimulation are efficiently transduced with modified lentiviral vectors carrying a GFP gene. The transduced cells fully reconstitute hematopoiesis in lethally irradiated mice and 10-15 percent of all cell lineages examined express GFP after 3 months. These results suggest that quiescent, hematopoietic stem cells are efficiently transduced by lentiviral vectors and that infection does not impair self renewal and lineage specification in stem cells which mediate long term reconstitution of lethally irradiated animals. Since the life span of sickle red cells is significantly shorter than normal., we speculate that correction of 10-15 percent of erythroid precursors in the marrow will translate to a major fraction in the peripheral blood because genetically corrected erythrocytes have a selective survival advantage. The anti-sickling gene we have developed will significantly reduce morbidity and mortality if the gene is expressed at 10 percent of betaS. If expression of the transduced gene is low, we propose to introduce a modified transcription factor (delta-EKLF) that will efficiently activate the human delta-globin gene. We have demonstrated that low levels of a modified EKLF activate the delta-globin gene at high levels, and HbA2 (alpha2 delta2) has powerful anti-sickling properties.